An off-line high pH reversed-phase fractionation and nano-liquid chromatography–mass spectrometry method for global proteomic profiling of cell lines

• High pH reversed-phase fractionation (RPF)-nano-LC–MS/MS method was developed.
• The method was applied in the global proteomic profiling of six cell lines.
• 7300–8956 proteins were identified in the cell samples.
• Showing greater advantages in the number and shorter time.
• RPF-nano-LC–MS/MS achieved an extensive proteomic coverage of cell lines.

Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) and first-dimensional fractionation is widely used for reducing sample complexity in large-scale proteomic profiling experiments. However, the limited number of proteins identified and the relatively long running time are a barrier to the successful application of this approach. In this study, off-line high pH reversed-phase fractionation (RPF) was combined with nano-LC–MS/MS in order to develop an improved method for global proteomic profiling of different cell lines. In the first dimensional reverse phase HPLC separation, 300 μg of digested cell protein was separated into 78 fractions under high pH conditions and condensed into 26 fractions for the second nano-LC–MS/MS analysis at low pH. The chromatographic conditions for the first and second steps were optimized, and the accuracy and reproducibility of protein quantification were investigated with an average Pearson correlation coefficient of 0.94. The method was then applied in the identification of proteins in six common cell lines (DMS, MFM, HepG2, U2OS, 293T and yeast), which resulted in identification of 7300–8500 and 8956 proteins in heavy/light labeled and label-free cell samples, respectively, in 1.5 days. The performance of the developed method was compared with isoelectric focusing (IEF)-nano-LC–MS/MS and the previously reported method; and off-line high pH RPF-nano-LC–MS/MS proved advantageous in terms of the number of proteins identified and the analytical time needed to achieve a successful global proteomic profiling outcome. The RPF-nano-LC–MS/MS method identified more proteins from low abundance (150 μg) samples with an average sequence coverage for each cell line of 23.4–35.1%. RPF-nano-LC–MS/MS may therefore be an efficient alternative tool for achieving improved proteomic coverage of multiple cell lines.